Expression of the renin-angiotensin system (RAS) in metanephric organ culture: evidence of an avascular RAS.

In order to determine the role of angiotensin II (AII) and its receptors in the control of early renal morphogenesis, an in vitro model of whole organ culture has been utilized to study the development of the RAS in culture. Tubular and glomerular differentiation occurs in the absence of a vascular network when metanephroi from E14 rat fetuses, composed only of mesenchyme and ureteric epithelia, are cultured for 6 days in chemically-defined, serum-free media. RT-PCR was used to determine the presence of mRNA for renin and the AII receptors (AT-1 and AT-2) at E14 and following in vitro development (X6). In situ hybridization localized renin mRNA in the differentiated explant. Immunohistochemistry was used to localize renin protein and radioligand binding was used to localize AT-1 and AT-2 receptors. Tissue AII content was determined by RIA. Renin mRNA was detected at E14 and a few large cells scattered throughout the mesenchyme contained renin protein. At X6, renin mRNA was localized to the branching ureteric epithelium. Renin protein was found in the branches of the ureteric bud, tubules, and developing glomeruli. At E14 both AT-1 and AT-2 receptors were found within the metanephros. 80% of the receptors were AT-2, and both receptor subtypes were localized to the metanephric mesenchyme. After culture, expression of both receptors was maintained although AT-1 expression had increased, mimicking the pattern in vivo. Metanephric AII content increased from 1.02±0.16 pg/kidney (0.10 pg/μg protein) to 7.08±0.63 pg/kidney (0.16 pg/μg protein) during culture. These results show that the components of the RAS are present in the metanephros at E14, prior to the development of renal vasculature, and that AII is actively produced. The localization and quantity of RAS components changes during in vitro differentiation. These results confirm the existence of a functional local RAS within the kidney. In addition, metanephric organ culture is a viable model in which to study the role of the RAS in nephrogenesis.